SIRT5 impairs aggregation and activation of the signaling adaptor MAVS through catalyzing lysine desuccinylation
2020; Springer Nature; Volume: 39; Issue: 11 Linguagem: Inglês
10.15252/embj.2019103285
ISSN1460-2075
AutoresXing Liu, Chunchun Zhu, Huangyuan Zha, Jinhua Tang, Fangjing Rong, Xiaoyun Chen, Sijia Fan, Chenxi Xu, Juan Du, Junji Zhu, Jing Wang, Gang Ouyang, Guangqing Yu, Xiaolian Cai, Zhu Chen, Wuhan Xiao,
Tópico(s)Toxoplasma gondii Research Studies
ResumoArticle17 April 2020free access Source DataTransparent process SIRT5 impairs aggregation and activation of the signaling adaptor MAVS through catalyzing lysine desuccinylation Xing Liu orcid.org/0000-0001-9338-7440 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Chunchun Zhu State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Huangyuan Zha State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Jinhua Tang State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Fangjing Rong State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiaoyun Chen State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Sijia Fan State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chenxi Xu State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Juan Du State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Junji Zhu State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jing Wang State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Gang Ouyang State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Guangqing Yu State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiaolian Cai State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Zhu Chen State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Wuhan Xiao Corresponding Author [email protected] orcid.org/0000-0002-2978-0616 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China The Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, Wuhan, China The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Xing Liu orcid.org/0000-0001-9338-7440 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Chunchun Zhu State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Huangyuan Zha State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Jinhua Tang State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Fangjing Rong State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiaoyun Chen State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Sijia Fan State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Chenxi Xu State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Juan Du State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Junji Zhu State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Jing Wang State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Gang Ouyang State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Guangqing Yu State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Xiaolian Cai State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Zhu Chen State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Wuhan Xiao Corresponding Author [email protected] orcid.org/0000-0002-2978-0616 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China The Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, Wuhan, China The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Author Information Xing Liu1,‡, Chunchun Zhu1,2,‡, Huangyuan Zha1, Jinhua Tang1,2, Fangjing Rong1,2, Xiaoyun Chen1,2, Sijia Fan1,2, Chenxi Xu1, Juan Du1, Junji Zhu1,2, Jing Wang1, Gang Ouyang1, Guangqing Yu1,2, Xiaolian Cai1, Zhu Chen1 and Wuhan Xiao *,1,2,3,4,5 1State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China 2University of Chinese Academy of Sciences, Beijing, China 3The Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, Wuhan, China 4The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China 5The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 27 68780087; Fax: +86 27 68780087; E-mail: [email protected] EMBO J (2020)39:e103285https://doi.org/10.15252/embj.2019103285 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract RLR-mediated type I IFN production plays a pivotal role in innate antiviral immune responses, where the signaling adaptor MAVS is a critical determinant. Here, we show that MAVS is a physiological substrate of SIRT5. Moreover, MAVS is succinylated upon viral challenge, and SIRT5 catalyzes desuccinylation of MAVS. Mass spectrometric analysis indicated that Lysine 7 of MAVS is succinylated. SIRT5-catalyzed desuccinylation of MAVS at Lysine 7 diminishes the formation of MAVS aggregation after viral infection, resulting in the inhibition of MAVS activation and leading to the impairment of type I IFN production and antiviral gene expression. However, the enzyme-deficient mutant of SIRT5 (SIRT5-H158Y) loses its suppressive role on MAVS activation. Furthermore, we show that Sirt5-deficient mice are resistant to viral infection. Our study reveals the critical role of SIRT5 in limiting RLR signaling through desuccinylating MAVS. Synopsis MAVS is the central molecule that links viral recognition to downstream antiviral kinase cascade and post translational modifications (PTMs) are important regulators of MAVS function. Here, molecular and functional characterization reveals that desuccinylation of MAVS by SIRT5 are critical for the modulation of MAVS activation. MAVS is a physiological substrate of SIRT5. MAVS is succinylated upon viral challenge. SIRT5 catalyzes desuccinylation of MAVS at Lysine 7. Desuccinylation of MAVS by SIRT5 diminishes the formation of MAVS aggregation, resulting in the inhibition of MAVS activation. Introduction Innate immunity, as the first line of defense against infection, senses pathogens, including RNA and DNA viruses, and subsequently triggers immune cells to secrete cytokines through germline-encoded pattern recognition receptors (PRRs; Chen & Ichinohe, 2015; Tan et al, 2018). During RNA viral infection, cytosolic RNA species are recognized by retinoic acid-inducible (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5; Moore & Ting, 2008; Wu & Chen, 2014; Brubaker et al, 2015; Roers et al, 2016). The activation of RIG-I and MDA5 leads to the recruitment of the signaling adaptor protein MAVS (also referred to as IPS-1, VISA, or Cardif) to activate the downstream protein kinase TBK1, resulting in phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) to drive type I IFN production, a family of cytokines that are essential for host protection against viral infection (Kawai et al, 2005; Meylan et al, 2005; Seth et al, 2005; Xu et al, 2005; Xie et al, 2012; Tan et al, 2018). Increasing attention has been paid to post-translational modifications (PTMs) of innate sensors and downstream signaling molecules, which influence their activity and function by inducing their covalent linkage to new functional groups (Liu et al, 2015; Liu et al, 2016a; Tan et al, 2018). Kdm6a promotes IL-6 expression through demethylating H3K27me3 at promoter of IL-6 (Li et al, 2017). HDAC9 deacetylates TBK1 for the activation of antiviral innate immunity (Li et al, 2016). HDAC6 modulates viral RNA sensing by deacetylating RIG-I (Choi et al, 2016). Deamidation of cGAS by HSV UL37 protein enhances viral replication (Zhang et al, 2018). In addition, viral homologs of phosphoribosylformylglycinamidine synthase (PFAS) recruit cellular PFAS to deamidate and activate RIG-I (He et al, 2015). As a critical adaptor protein in RLR signaling, the normal function of MAVS has been shown to be markedly influenced by its ubiquitination (You et al, 2009; Liu et al, 2017; He et al, 2019), phosphorylation (Liu et al, 2015), and O-GlcNAcylation (Li et al, 2018a). Recently, more other modifications of MAVS that influence antiviral innate immunity by modulating MAVS function have been identified (Liu & Gao, 2018; Yang et al, 2019). Accumulating evidence suggests that intermediates and derivatives of the Krebs cycle and glucose metabolism possess non-metabolic signaling functions, such as regulation of cellular immunity, in addition to its traditional functions associated with bioenergetics or biosynthesis (Li et al, 2018a; Mills et al, 2018b; Williams & O'Neill, 2018; Ryan et al, 2019; Zhang et al, 2019). Krebs cycle-derived metabolites have been shown to attribute both signaling functions and impact on multiple processes critical for the cellular immune response. These metabolites can either act as immune signaling molecules to inhibit specific enzymes or drive modification of proteins in the immune signaling pathway (Rubic et al, 2008). As the only metabolite directly linking the Krebs cycle and the mitochondrial respiratory chain, succinate acts as a signaling molecule to exert its inflammatory effects through several pathways (Rubic et al, 2008; Tannahill et al, 2013; Littlewood-Evans et al, 2016; Lei et al, 2018; Peruzzotti-Jametti et al, 2018). Recently, the critical role of succinate in autoimmune and autoinflammatory has been recognized (Tannahill et al, 2013, 2013; Littlewood-Evans et al, 2016; Mills et al, 2016, 2018a; Li et al, 2018b). Dysregulation of succinate metabolism can cause accumulation of succinyl-CoA, which may induce lysine succinylation (Peyssonnaux et al, 2007; Zhang et al, 2011; Xie et al, 2012; Park et al, 2013; Weinert et al, 2013). The enzyme responsible for succinylation has yet to be identified, and indeed, it is possible that this reaction occurs non-enzymatically by direct reaction between succinyl-CoA and the modified protein (Feng et al, 2017). It is evident that succinylation can modulate macrophage function. Succinylation of Lys311 of pyruvate kinase M2 (PKM2), a key glycolytic enzyme required for the shift to glycolysis in activated macrophages, has been shown to limit its activity by promoting its tetramer-to-dimer transition (Wang et al, 2017a). However, whether succinylation of innate immune sensors and downstream signaling molecules exists or succinylation influences innate immunity in response to viral infection is still largely unknown. Sirtuin 5 (SIRT5) belongs to the sirtuin family of NAD+-dependent deacetylases (Finkel et al, 2009). The deacetylase activity of SIRT5 is barely detected (Anderson et al, 2014). Of note, SIRT5 can promote acetylation of p65 through blocking the deacetylation of p65 catalyzed by SIRT2 in a deacetylase activity-independent manner (Qin et al, 2017). In fact, SIRT5 has been shown to have potent desuccinylase activity (Finkel et al, 2009; Du et al, 2011; Park et al, 2013; Rardin et al, 2013; Tannahill et al, 2013; Wang et al, 2017a). Interestingly, LPS decreases SIRT5 expression in macrophages and increases protein succinylation (Tannahill et al, 2013). Consistent with the activation of PKM2 by SIRT5-catalyzed desuccinylation, SIRT5-deficient mice exhibit hypersuccinylation and increased IL-1β production (Wang et al, 2017a). However, it has also been reported that Sirt5 deficiency does not compromise innate immune response to bacterial infections (Heinonen et al, 2018). Due to lack of convincing data to support the enzymes responsible for succinylation existed (Weinert et al, 2013; Feng et al, 2017; Yang & Gibson, 2019), in order to address whether succinylation influences innate immunity in response to viral infection, we examined the impact of SIRT5, a well-defined desuccinylase (Du et al, 2011; Park et al, 2013; Rardin et al, 2013; Anderson et al, 2014; Wagner & Hirschey, 2014; Wang et al, 2017a), on antiviral innate immunity. We found that SIRT5 negatively regulates the innate immunity in response to RNA viral infection. Further investigation shows that SIRT5 mediates desuccinylation of Lys7 of MAVS, leading to impairment of aggregation and activation of MAVS. These findings suggest a critical role of SIRT5 in limiting RLR signaling through desuccinylating MAVS. Results SIRT5 suppresses the MAVS-mediated RLR signaling To investigate whether SIRT5 participates in regulating RLR signaling, we employed a promoter assay to examine the effect of SIRT5 on promoter activity. IFNβ promoter luciferase reporter and ISRE-luciferase reporter (containing interferon stimulated response elements) are well-defined reporters for monitoring RLR activation (Xu et al, 2005; Zhong et al, 2008; Liu et al, 2016b). Upon overexpression of SIRT5 in HEK293T cells or H1299 cells, Sendai virus (SeV)-induced IFNβ promoter reporter (IFNβ-luc.) activity and ISRE-luciferase reporter activity were strongly inhibited (Fig 1A–D). Consistent with these observations, SeV-induced IFNβ promoter reporter activity and ISRE-luciferase reporter activity were enhanced upon SIRT5 knockdown by small interfering RNAs (si-SIRT5#1 and si-SIRT5#2; Wang et al, 2017a) in H1299 cells (Fig 1E and F). To further confirm the suppressive role of SIRT5 on RLR signaling, we knocked out SIRT5 in H1299 cells via CRISPR/Cas9. Consistently, SeV-induced IFNβ promoter reporter activity and ISRE-luciferase reporter activity were enhanced in SIRT5−/− cells compared to the wild-type H1299 cells (SIRT5+/+; Fig 1G and H). In addition, poly(I:C) (RNA virus mimics)-induced ISRE-luciferase reporter activity was suppressed by the overexpression of SIRT5 in a dose-dependent manner (Fig 1I). These findings indicate that SIRT5 inhibits RLR signaling. Figure 1. SIRT5 suppresses the MAVS-mediated type I IFN signaling A, B. IFNβ promoter activity (A) and ISRE reporter activity (B) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected HEK293T cells with or without SeV infection (SeV or UI) for 18–24 h. C, D. IFNβ promoter activity (C) and ISRE reporter activity (D) in Myc empty vector (200 ng) or Myc-SIRT5 (200 ng)-transfected H1299 cells with or without SeV infection (SeV or UI) for 18–24 h. E, F. IFNβ promoter activity (E) and ISRE reporter activity (F) in the indicated siRNA-transfected H1299 cells (si-NC, si-SIRT5#1, and si-SIRT5#2) with or without SeV infection (SeV or UI) for 18–24 h. NC, negative control. G, H. IFNβ promoter activity (G) and ISRE reporter activity (H) in SIRT5-deficient H1299 cells (SIRT5−/−) or the wild-type (WT) H1299 cells (SIRT5+/+) with or without SeV infection (SeV or UI) for 18–24 h. I. ISRE reporter activity in Myc-SIRT5 (0, 100, or 200 ng)-transfected HEK293T cells with or without poly(I:C) transfection for 18–24 h. J. ISRE reporter activity in co-transfection of Myc-RIG-I (200 ng) together with Myc-SIRT5 (0, 100, or 200 ng)-transfected HEK293T cells for 24 h. K. ISRE reporter activity in co-transfection of Myc-MDA5 (200 ng) together with Myc-SIRT5 (0, 100, or 200 ng) in HEK293T cells for 24 h. L. ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (0, 100, or 200 ng) in HEK293T cells for 24 h. M. ISRE reporter activity in co-transfection of Myc-TBK1 (200 ng) together with Myc-SIRT5 (0, 100, or 200 ng) in HEK293T cells for 24 h. N. ISRE reporter activity in co-transfection of HA-IRF3 (200 ng) together with Myc-SIRT5 (0, 100, or 200 ng) in HEK293T cells for 24 h. O. ISRE reporter activity in co-transfection of HA-MAVS (200 ng) together with Myc-SIRT5 (200 ng) or Myc-SIRT5-H158Y (200 ng), respectively, in HEK293T cells for 24 h. Data information: Graphs represent fold induction relative to the luciferase activity in the control cells. UI, uninfected. All data are presented as the mean values based on three independent experiments, and error bars indicate s.e.m. (unpaired two-tailed Student's t-test). Download figure Download PowerPoint Subsequently, we sought to determine which target in RLR signaling mediates SIRT5's suppressive function. The ISRE-luciferase reporter assay revealed that overexpression of SIRT5 caused a reduction of luciferase activity, which was induced by RIG-I, MDA5, or MAVS in a dose-dependent manner (Fig 1J–L). However, overexpression of SIRT5 showed no effect on luciferase activity driven by TBK1, or IRF3, suggesting that SIRT5 functions at the MAVS level (Fig 1M and N). Of note, overexpression of enzyme-deficient mutant of SIRT5 (SIRT5-H158Y; Nakagawa et al, 2009; Zhang et al, 2017) had no effect on luciferase activity driven by MAVS, in contrast to overexpression of WT SIRT5 (Fig 1O). Expressions of the transfected plasmids, the efficacy of SIRT5-knocked down by siRNA and SIRT5-knocked out by CRISPR/Cas9 were confirmed by Western blot analysis (Appendix Fig S1A–O). To further validate these results, we performed dose-titration assays. Overexpression of SIRT5 suppressed dose-dependent activation of IFNβ promoter activity by SeV infection in HEK293T cells (Appendix Fig S2A). Overexpression of SIRT5 also suppressed dose-dependent activation of ISRE-luciferase reporter activity by transfection of increasing amount of MDA5 in HEK293T cells (Appendix Fig S2B). However, overexpression of SIRT5 had no effect on dose-dependent activation of ISRE reporter activity by transfection of increasing amount of IRF3 in HEK293T cells (Appendix Fig S2C). Protein expressions of transfected plasmids were confirmed by Western blot analysis (Appendix Fig S2D–F). Of note, overexpression of SIRT5 did not influence the ISRE-luciferase reporter activity induced by co-transfection of cGAS and STING, suggesting that SIRT5 might have no effect on the cytosolic DNA sensing pathway (Tan et al, 2018; Appendix Fig S2G). In addition, overexpression of SIRT2 did not affect ISRE-luciferase reporter activity and IFNβ promoter activity induced by transfection of MAVS, indicating that SIRT5 might be the specific one in the sirtuin family to inhibit MAVS function (Appendix Fig S2H and I; Finkel et al, 2009). Protein expressions of transfected plasmids were confirmed by Western blot analysis (Appendix Fig S2J–L). Taken together, these results suggest that SIRT5 regulates RLR signaling by influencing MAVS function. SIRT5 interacts with MAVS The observations that SIRT5 suppresses the activation of MAVS on RLR signaling prompted us to determine whether SIRT5 influences MAVS's function through protein–protein interaction. First, we examined their co-localization by immunofluorescence staining using anti-MAVS and anti-SIRT5 antibodies. Fluorescence confocal microscopy showed that SIRT5 co-localized with MAVS in the mitochondria in both H1299 cells and HeLa cells (Fig 2A, Appendix Figs S3A and B, and S4A–D; Anderson et al, 2014; Liu et al, 2017). Co-immunoprecipitation assays showed that ectopically expressed SIRT5 pulled down ectopically expressed MAVS in HEK293T cells and vice versa (Fig 2B and C). In H1299 cells, endogenous MAVS was also co-immunoprecipitated with endogenous SIRT5 (Fig 2D), while endogenous co-immunoprecipitation between MAVS and SIRT5 was not detected in SIRT5-deficient H1299 cells (SIRT5−/−; Fig 2E). Escherichia coli-expressed GST-tagged MAVS interacted with E. coli-expressed His-tagged SIRT5 in vitro (Fig 2F). These data indicated that SIRT5 directly associated with MAVS. Further domain mapping of MAVS binding to SIRT5 indicated that the TM domain of MAVS was required for SIRT5 interaction (Fig 2G–I). However, SIRT5 did not influence protein stability of MAVS revealed by either overexpression of SIRT5 in HEK293T cells or knockout of SIRT5 in H1299 cells (Appendix Fig S5A and B). Figure 2. SIRT5 interacts with MAVS A. Confocal microscopy image of endogenous SIRT5 co-localized with endogenous MAVS in H1299 cells detected by immunofluorescence staining using anti-SIRT5 and anti-MAVS antibodies. Mito, MitoTracker; Scale bar = 8 μm. B, C. Co-immunoprecipitation of Myc-SIRT5 with HA-MAVS and vice versa. HEK293T cells were co-transfected with indicated plasmids for 24 h. Anti-Myc (B) or anti-HA antibody-conjugated agarose beads (C) were used for immunoprecipitation, and the interaction was detected by immunoblotting with the indicated antibodies. D. Endogenous interaction between MAVS and SIRT5. Anti-MAVS antibody was used for immunoprecipitation, and normal mouse IgG was used as a control. E. Endogenous interaction between MAVS and SIRT5 in the wild-type (WT) (MAVS+/+) or SIRT5-deficient (MAVS−/−) H1299 cells. Anti-MAVS antibody was used for immunoprecipitation, and the interaction was detected by immunoblotting with anti-SIRT5 antibody. F. GST pull-down assay for GST-tagged MAVS and His-tagged SIRT5. GST-tagged MAVS and His-tagged SIRT5 were expressed in Escherichia coli (BL21), respectively. The association of GST-MAVS with His-SIRT5 was detected by immunoblotting with anti-SIRT5 antibody. GST and GST-MAVS proteins were stained with Coomassie blue. G. Schematic of MAVS domains interacted with SIRT5. The interaction is indicated by plus (+) sign. H, I. Co-immunoprecipitation analysis of HA-SIRT5 with Flag-MAVS-truncated mutants. HEK293T cells were co-transfected with the indicated plasmids. Anti-Flag antibody-conjugated agarose beads were used for immunoprecipitation, and the interaction was analyzed by immunoblotting with the indicated antibodies. Flag-MAVS fragments (WT: full length; ΔC1, 1–173 aa; ΔC2, 1–513 aa; ΔN1, 91–540 aa; ΔN2, 173–540 aa; ΔCARD, Δ10–77 aa; ΔPR, Δ103–173 aa; ΔTM, Δ514–535 aa). Data information: IP, immunoprecipitation; TCL, total cell lysates. Source data are available online for this figure. Source Data for Figure 2 [embj2019103285-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint SIRT5 desuccinylates Lysine 7 of MAVS Given a well-defined function of SIRT5 in desuccinylation, we sought to determine whether SIRT5 could influence MAVS succinylation (Du et al, 2011; Park et al, 2013; Rardin et al, 2013; Anderson et al, 2014; Wagner & Hirschey, 2014; Wang et al, 2017a). Initially, we examined whether succinylation of MAVS existed. Using in vitro succinylation assays, we found that MAVS expressed in either E. coli or HEK293T cells could be readily succinylated by adding succinyl-CoA in a dose-dependent manner (Fig 3A and B), suggesting that MAVS could be succinylated. Figure 3. MAVS is succinylated at Lysine 7, and SIRT5 mediates its desuccinylation A, B. MAVS could be succinylated in vitro. GST-MAVS (A) extracted from Escherichia coli or Flag-tagged MAVS (B) protein purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. Protein succinylation was detected with anti-pan-succinyl-lysine antibody; GST-MAVS or Flag-MAVS was stained by Coomassie blue. C. The succinylated residue in MAVS was identified by mass spectrometry analysis. D. Sequence alignment of partial MAVS (1–30 amino acids) from human, macaque, cow, pig, dog, mouse, and rat. The red box indicates a conserved lysine (K7). E. Dot blot assay for the specificity of anti-succ-K7-MAVS antibody. Equal amounts of succinyl-peptides or the control peptides were immunoblotted with the indicated dilutions of anti-succ-K7-MAVS antibody. F, G. GST-MAVS (F) extracted from E. coli or Flag-MAVS (G) purified from HEK293T cells was incubated with the indicated concentrations of succinyl-CoA. The succinylation of MAVS at Lysine 7 was detected by anti-succ-K7-MAVS antibody. H. Disruption of SIRT5 in H1299 cells enhanced succinylation of MAVS at Lys 7 compared to that in the SIRT5-intact H1299 cells (SIRT5+/+) (3.2 vs. 1.0). The cell lysates from SIRT5-deficient H1299 cells or the SIRT5-intact H1299 cells were immunoprecipitated with anti-MAVS antibody or mouse IgG control, followed by immunoblotting with anti-succ-K7-MAVS antibody. I. Reconstitution of WT SIRT5 in SIRT5-deficient H1299 cells (SIRT5−/−) caused a significant reduction in succinylation of MAVS at Lys 7 (1.0 vs. 0.2); but overexpression of SIRT5-H158Y in SIRT5−/− H1299 cells has no effect on the reduction in succinylation of MAVS at Lys 7 (1.0 vs. 1.2). The SIRT5−/− H1299 cells were transfected with Flag-SIRT5 or Flag-SIRT5-H158Y, followed by immunoprecipitating with anti-MAVS antibody or mouse IgG control, and immunoblotting with anti-succ-K7-MAVS antibody. J, K. Knockout of Sirt5 increased MAVS succinylation in mouse livers (J) and lungs (K). Proteins extracted from livers (J) and lungs (K) of Sirt5 KO and the wild-type littermates (n = 3 per group) were detected by the indicated antibodies. MAVS succinylation was determined by anti-succ-K7-MAVS antibody, and anti-pan-succinyl-lysine antibody was used as positive controls. Data information: IP, immunoprecipitation; TCL, total cell lysates. Source data are available online for this figure. Source Data for Figure 3 [embj2019103285-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Subsequently, we examined the succinylation site (s) on MAVS through mass spectrometry (MS) analysis. One succinylation site (Lysine 7) in MAVS was identified (Fig 3C). Lysine 7 of MAVS is evolutionary conserved across species (Fig 3D). To further confirm this succinylated site in MAVS, we d
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